Project Summary/Abstract RNA modifications are numerous, and range from simple methylation to complex changes such as addition of aminoacylcarbamoyl moieties. These modifications and, the levels at which are present, are emerging as important regulators of biological processes, and perturbations such as chemical or drug exposure can alter the patterns of specific modifications on RNA sub-types. Moreover, given their variety, specificity and their relationship to function and structure stability, RNA modifications are potentially valuable biomarkers of disease and treatment progression, especially given that modified nucleosides are relatively stable even after RNA degradation, and can be released from cells into bodily fluids. The long term objective of this study is to facilitae research into RNA modifications and their impact on health and disease. The immediate hurdle, and the goal of this application, is to develop extremely sensitive analytical methods for detection and quantification of common and novel RNA modifications that can be readily disseminated to the RNA research community. The test case biological system is based on human induced pluripotent stem cell derived nervous system cells that will be exposed to two different drugs of abuse, nicotine and cannabinoids. Aim 1. We will use RNA modified nucleoside standards to develop highly sensitive and robust method of analysis. The ultimate tool we will be developing is a product resultant from the combination of two technologies, UHPLC and MS that will be optimized for high resolution separation and highly sensitive detection of RNA modifications. The tools developed will be tested on differentiated anterior cerebral cortex neurons and glia cells and RNA modifications will be characterized and abundances quantified during the different stages of maturation. Aim 2. Tools developed during aim 1 will be further adapted to detect specific RNA modifications that may be playing a significant role on differentiated forebrain cells, as a result from treatment with drug of abuse. During this aim we will specifically study forebrain cells that will be exposed to different nicotie and cannabinoids concentrations and compared to vehicle controls. The results of the methods we are proposing will be in the form of highly sensitive and accurate tools for the analysis of a wide range of chemistries and abundances of RNA modifications present in regulatory RNAs. The ultimately goal is to develop highly sensitive RNA modifications-based biomarkers discovery tools that requires minimal level of technical expertise as a way to disseminate and accelerate the field of RNA modifications with regards to gain further insight into mechanisms of disease.